Certain chemical and biological reactions are comprised of one or more intermediate steps where a reaction component is introduced for coupling with a suspected analyte contained in a sample such as blood or urine. Un-reacted portions of this intermediate reagent must be removed prior to additional steps in the reaction or prior to final detection or extraction of the analyte of interest.
For example, a fluorescence generating reagent may be added to reaction products to enable photometric detection of a specific analyte. This detection reagent is designed to bind to the analyte of interest. Detection reagent that has coupled with the complex of interest will yield a photometric signal in direct proportion to the quantity of complex present only if unbound fluorescence generating reagent is removed or otherwise deactivated. Free flouresence generating reagent that is not removed constitutes interference or background noise in the signal detection process and may lead to false positives or inaccurate quantitative analytical results.
Current immunoassay analysis contains many such reactions where one or more chemical components must be removed while preserving the integrity and quantitative accuracy of the original species of interest. Several techniques have evolved to assure complete removal of unwanted components in a cycle generally termed a wash cycle.
One such technique is to coat the inner surfaces of a container or well, such as a microtiter plate, with an antibody or antibody complex selected to bind to an analyte of interest. Blood or other sample is placed in the well. The analyte, if present in the sample, will combine with the complex predisposed on the container surface. A wash step follows to remove unreacted sample. A typical wash process consists of removal of liquid by an aspirating probe followed by the addition and removal of a rinse solution. Additional chemical components such as a radioactive label or a fluorescent tag may be introduced, reacted and removed by repeating this basic wash cycle. The original analyte remains bound to the container wall and available for reaction with subsequent reagent throughout all washing steps.
Immunoassay and other similar binding assays can be very sensitive, detecting extremely minute quantities of an analyte. An interfering material remaining from an incomplete wash may render the test inaccurate. Wash efficiency is thus an extremely important part of such analytical techniques.
Wash efficiency is governed by the quantity of unwanted, unbound material remaining after removal of the supernatant, the volume of rinse added, the quantity of desired, bound material that is inadvertently removed and the total number of wash cycles required. The quantity of unbound material remaining after n number of wash cycles may be expressed by (Z/Q).sup.n, where Z is the volume remaining after decanting and Q is the volume of rinse solution added. To achieve residuals in the nanogram range Z must be very small, Q maximized or the number of wash cycles, n, must be increased.
Increasing n, the number of cycles, has the disadvantage of slowing analytical throughput and increasing the probability of loss of desired, bound material. Wash cycles are time consuming and detract from processing speed in automated analytical instruments. Washing consumes significant quantities of rinse fluid which may contain expensive chemicals. Excessive or over vigorous washing may detach and remove or damage the analyte of interest. Expensive mechanisms and precisely molded containers are required to accurately place aspirating probes to achieve the lowest possible residue after decanting.
The above described coated container is reasonably efficient at washing contained volumes on the order of 100 microliters. Two significant limitations exist with coated containers. The first limitation is one of sensitivity due to the limited surface area available for coating. Attempts to increase surface area by roughening or providing other protruding features generally come at the expense of increasing the residual volume, Z, remaining after a wash decanting. The second limitation involves the volume of the container which serves to limit the rinse volume applicable at each wash cycle.
Another popular separation technique utilizes coated magnetic particles. Here small magnetic particles are coated with a material that is capable of binding an intermediate coupling agent such as antibodies. A reagent solution is comprised of coated particles coupled to specific antibodies disposed in a liquid carrier. A magnet is used to retain bound particles from loss during the rinse portion of a wash cycle. Magnetic particles offer improved sensitivity and speed of reaction due to the large available surface area when compared to coated containers. Particles pose no specific limitation on the volume of rinse cycle. Certain problems, however, exist with magnetic particles during a wash process. These problems include the extended time required to draw magnetic particles out of suspension by application of magnetic force, the magnetic particle collected mass or clumps tend to retain excessive fluid, and the clumped mass is difficult to re-suspend into solution.
A finite time is required to draw particles from suspension into a clumped mass necessary before decanting of rinse fluid can begin. The clumped mass generally remains wet with trapped unbound material effectively increasing the volume Z of unwanted material left behind on each wash cycle. One solution is to agitate and break up the clump in the presence of rinse fluid to improve the removal of unbound material on the next extraction--decant cycle. The problem with re-suspension is the time involved to both suspend and again magnetically attract the suspended particles. There is also increased risk of de-coupling and loosing analyte during such re-suspension efforts.
Magnetic particles differ in magnetohydrodynamic characteristics from lot to lot and within the same sample, thus, some percentage of particles do not magnetically extract in the time allotted for extraction and are lost in the rinse decant. The binding strength of the coating to the particle and the coating to the analyte is finite, restricting the intensity of fluid addition and re-suspension agitation to below levels that tend to exceed said binding strength.
The combination of extra washes required to remove unbound material with losses of some bound material on each wash cycle leads to quantitative errors using this type of assay. The hydrodynamic characteristics of particles, the hydrodynamic characteristics of particles bound to analyte and the precision of the wash cycle must all be carefully controlled to achieve a trustworthy wash process.
Magnetic particle technology continues to advance in performance of the particles and in the wide variety of substances available for binding, capture and extraction.
Yet another method of separation is described by Babson et. al. in U.S. Pat. No. 5,098,845. This patent describes a circular vessel containing a small sphere to which antibodies are attached. Wash separation is effected by rotating the cup about its longitudinal axis where centrifugal force serves to remove the liquid contents while the sphere remains in place. This approach is quite effective in rapidly removing all but the smallest traces of unbound material. Rinse water can be added while the container is spinning to remove the last traces of unbound material without stopping for re-suspension as in the case of magnetic particle separation. The method of Babson et al. has disadvantages similar to coated containers in that the surface area available for binding is limited to the dimension of the sphere.